We will purify the geranylgeranyltransferase (GGTase-II) that isoprenylates Ypt1 and Sec4 as well as define the domains of these GTP- binding proteins that interact with this enzyme. A ligand has been added onto a subunit of the transferase to facilitate the purification of this enzyme complex by affinity chromatography. The purified subunits will be cloned using a variety of molecular approaches, and the cloned genes will be mutagenized in vitro. The phenotypic consequences of mutagenizing each subunit of the GGTase-II will also be analyzed. Additionally, we are characterizing a suppressor (BTS1) of a mutant that is defective in one of the subunits of the transferase. BTS1 may encode a component of the GGTase-II or another prenyltransferase that acts on this pathway. Understanding the specificity of the GGTase-II, with respect to the farnesyltransferase and GGTase-I, may have therapeutic value in the treatment of cancer and heart disease. After Ypt1 is modified by the GGTase-II, it binds to membranes where it interacts with other proteins that function in conjunction with it to mediate a late stage in ER to Golgi transport. Currently, we are characterizing two membrane proteins, whose overexpression compensates for the loss of Ypt1 function. Crosslinking studies will be performed to determine if these proteins interact with the prenylated form of this GTP-binding protein. In addition, we have devised a genetic screen to identify new genes that encode the accessory proteins that interact with Ypt1. Ultimately, these studies will elucidate the role that this small GTP-binding protein plays in mediating vesicular traffic between the ER and Golgi complex.